Living organisms are under constant assault by mutagenic agents, including radiation from various sources and a wide range of chemicals in the environment. Cell survival is dependent upon effective repair of DNA damage caused by these agents. The most lethal form of DNA damage induced by these agents is double-strand breaks (DSBs) and, in the absence of DSB repair, cells die rapidly. However, some DSB repair pathways are dangerous because they can lead to genomic instability, resulting in chromosomal rearrangements. Chromosomal rearrangements have been implicated in oncogene activation and are the hallmark of many cancers. Therefore, characterization of pathways that predispose cells to genetic instability is critical for the understanding of tumorigenesis and may help to elucidate targets for cancer prevention and treatment. The proposed research will investigate the mechanism of break-induced replication (BIR), a poorly understood DSB repair pathway, which can lead to genetic instability. The current BIR model suggests that the junction made between the invading broken strand and the undamaged molecule initiates DNA synthesis on the intact chromosome, thereby acting as an origin of replication. This can result in copying of hundreds of kilobases of DNA from the donor molecule while a large piece of the unrepaired, broken DNA is lost during the next round of replication. BIR has been suggested to play an important role in the repair of collapsed replication forks, and also in several cancer-related phenomena, including telomere maintenance in the absence of telomerase, loss of heterozygosity, and formation of non-reciprocal translocations. The relevance of BIR to mechanisms underlying tumorigenesis has made this model a critical starting point for many branches of research. However, the basic tenet of BIR - bona fide replication to repair breaks in DNA was deduced based on genetic data but has never been demonstrated directly by physical analyses of intermediates and/or the final products of this process. The objective of the proposed research is to use two powerful technologies, dynamic molecular combing coupled with fluorescent in situ hybridization and two- dimensional gel electrophoresis, to test the central hypothesis of BIR;i.e., that DSB repair is achieved by the assembly and progression of a replication fork. The proposed research will determine the structure of BIR intermediates and products in yeast Saccharomyces cerevisiae (a model genetic organism). Also, it will estimate the speed of BIR and its ability to replicate through centromeres and known barrier sites. In addition, the roles of individual proteins involved in BIR will be identified. The proposed approach is unique because, unlike other studies of BIR that rely only on indirect genetic or population physical data, it will enable visualization of BIR in individual DNA molecules using dynamic molecular combing, while two-dimensional gel electrophoresis will help to analyze replication fork intermediates.Narrative. The proposed research is aimed to determine the mechanism of break-induced replication (BIR) by using two powerful technologies: molecular combing and two-dimensional gel electrophoresis. This research will test the basic tenet of the BIR model, namely, the assembly of a bona fide replication fork to repair double-strand breaks in DNA. This knowledge is critical to further the understanding of several cancer-related phenomena, including telomere maintenance in the absence of telomerase, loss of heterozygosity, and formation of non-reciprocal translocations.